The program is divided into several parts. The first is related to issues associated with the safety of cell substrates. The second is directed to developing quantitative assays for adventitious agents. The third is concerned with understanding how HIV evolves during HIV disease, what coreceptors the virus uses to enter cells, and what are the determinants of tropism; this information will be relevant to the design of AIDS vaccines and therapeutics. One of the major issues that has yet to be resolved with respect to cell substrates that are either tumorigenic or are derived from tumors is whether residual cell-substrate DNA poses a safety risk to vaccine recipients. This risk could be from the oncogenic activity of the DNA, if the DNA contains activated dominant oncogenes (cellular of viral), or from its infectivity, if the DNA contains the genome of a DNA virus or the proviral copy of a retrovirus. Quantitative assays are being developed to be able to assess the risk for both these activities. An in vivo assay to detect inoculated cellular oncogenes in rodents is being developed. An in vitro assay to quantify infectious DNA of a retrovirus has been developed and has revealed that as little as 1 pg of cloned retroviral genome and 5 ?g of DNA from infected cells can be detected. Current work is directed towards determining methods to reduce DNA infectivity. With respect to adventitious agents, in collaboration with Andrew Lewis (DVP/OVRR/CBER), we have developed quantitative, real-time PCR assays for the primate polyomaviruses JCV, BKV, and SV40. Currently, Dr Lewis and I, in collaboration with Dr Ed Tabor (OBRR), are using the Q-PCR assays to investigate whether SV40, originally discovered as a contaminant in poliovirus and adenovirus vaccines, is a contaminant in the blood supply and, if so, at what level. BKV and JCV will serve as positive controls. We are also developing immunological assays to answer whether antibodies to SV40 are present in humans and whether these antibodies are neutralizing. For this, we have developed a reporter-gene based assay for SV40 and are developing analogous assays for BKV and JCV. With these neutralization assays, we will be able for the first time to determine the neutralization titers for the three polyomaviruses in human serum. We have initiated a small study to develop a neutralization assay for the SARS coronavirus. This will involve generating pseudotyped viruses and using antibodies purified from infected individuals. Our work on HIV has centered on determining what coreceptors HIV uses to enter various cells and whether HIV can adapt to use one of the minor coreceptors. Understanding how HIV adapts and to what receptor will assist in vaccine design and vaccine safety. We have identified a CCR5-using HIV strain that can use CXCR6 poorly for entry but that adapts on passage to use it well. As this receptor is expressed in placental tissue and in the thymus, the possibility exists that HIV uses CXCR6 in these tissues or that HIV adapts to use this receptor at certain stages in HIV disease. Importantly, additional passage in cells expressing both CXCR6 and CXCR4, one of the major coreceptors, results in the evolution of a virus that now can use CXCR4. Changes in the envelope genes are being determined. This project incorporates FY2002 projects 1Z01BK003011-09, 1Z01BK003012-09, and 1Z01BK003017-07.